Herbert Wertheim School of Optometry and Vision Science, University of California, Berkeley, CA, USA.
Vision Science Program, University of California, Berkeley, CA, USA.
Mol Neurodegener. 2024 Nov 20;19(1):86. doi: 10.1186/s13024-024-00775-z.
The resident astrocyte-retinal ganglion cell (RGC) lipoxin circuit is impaired during retinal stress, which includes ocular hypertension-induced neuropathy. Lipoxin B produced by homeostatic astrocytes directly acts on RGCs to increase survival and function in ocular hypertension-induced neuropathy. RGC death in the retina and axonal degeneration in the optic nerve are driven by the complex interactions between microglia and macroglia. Whether LXB neuroprotective actions include regulation of other cell types in the retina and/or optic nerve is an important knowledge gap.
Cellular targets and signaling of LXB in the retina were defined by single-cell RNA sequencing. Retinal neurodegeneration was induced by injecting silicone oil into the anterior chamber of mouse eyes, which induced sustained and stable ocular hypertension. Morphological characterization of microglia populations in the retina and optic nerve was established by MorphOMICs and pseudotime trajectory analyses. The pathways and mechanisms of action of LXB in the optic nerve were investigated using bulk RNA sequencing. Transcriptomics data was validated by qPCR and immunohistochemistry. Differences between experimental groups were assessed by Student's t-test and one-way ANOVA.
Single-cell transcriptomics identified microglia as a primary target for LXB in the healthy retina. LXB downregulated genes that drive microglia environmental sensing and reactivity responses. Analysis of microglial function revealed that ocular hypertension induced distinct, temporally defined, and dynamic phenotypes in the retina and, unexpectedly, in the distal myelinated optic nerve. Microglial expression of CD74, a marker of disease-associated microglia in the brain, was only induced in a unique population of optic nerve microglia, but not in the retina. Genetic deletion of lipoxin formation correlated with the presence of a CD74 optic nerve microglia population in normotensive eyes, while LXB treatment during ocular hypertension shifted optic nerve microglia toward a homeostatic morphology and non-reactive state and downregulated the expression of CD74. Furthermore, we identified a correlation between CD74 and phospho-phosphoinositide 3-kinases (p-PI3K) expression levels in the optic nerve, which was reduced by LXB treatment.
We identified early and dynamic changes in the microglia functional phenotype, reactivity, and induction of a unique CD74 microglia population in the distal optic nerve as key features of ocular hypertension-induced neurodegeneration. Our findings establish microglia regulation as a novel LXB target in the retina and optic nerve. LXB maintenance of a homeostatic optic nerve microglia phenotype and inhibition of a disease-associated phenotype are potential neuroprotective mechanisms for the resident LXB pathway.
在视网膜应激过程中，包括眼压升高引起的神经病，驻留的星形胶质细胞-视网膜神经节细胞（RGC）脂氧素回路受损。稳态星形胶质细胞产生的脂氧素 B 直接作用于 RGC，增加眼压升高诱导的神经病中的存活和功能。视网膜中 RGC 的死亡和视神经中的轴突变性是由小胶质细胞和大胶质细胞之间的复杂相互作用驱动的。LXB 的神经保护作用是否包括对视网膜和/或视神经中其他细胞类型的调节，这是一个重要的知识空白。
通过单细胞 RNA 测序确定 LXB 在视网膜中的细胞靶标和信号。通过在前房内注射硅油来诱导小鼠眼睛的神经退行性变，从而诱导持续和稳定的眼压升高。通过 MorphOMICs 和拟时轨迹分析建立视网膜和视神经中小胶质细胞群体的形态特征。使用批量 RNA 测序研究 LXB 在视神经中的作用途径和机制。通过 qPCR 和免疫组织化学验证转录组数据。通过 Student's t 检验和单向方差分析评估实验组之间的差异。
单细胞转录组学将小胶质细胞鉴定为健康视网膜中 LXB 的主要靶标。LXB 下调了驱动小胶质细胞环境感应和反应性的基因。小胶质细胞功能分析表明，眼压升高诱导了视网膜和出乎意料的远端有髓视神经中的独特、时间定义和动态表型。CD74 在视神经中仅诱导独特的小胶质细胞群体，但在视网膜中不诱导小胶质细胞的表达，CD74 是大脑中与疾病相关小胶质细胞的标志物。在正常眼压下，脂氧素形成的基因缺失与存在 CD74 视神经小胶质细胞群体相关，而在眼压升高期间，LXB 治疗使视神经小胶质细胞向稳态形态和非反应状态转变，并下调 CD74 的表达。此外，我们发现 CD74 与视神经中磷酸化磷酯酰肌醇 3-激酶（p-PI3K）表达水平之间存在相关性，LXB 治疗降低了这种相关性。
我们确定了小胶质细胞功能表型的早期和动态变化、在远端视神经中诱导独特的 CD74 小胶质细胞群体，这些都是眼压升高诱导的神经退行性变的关键特征。我们的发现确立了小胶质细胞调节作为视网膜和视神经中新型 LXB 靶标。LXB 维持视神经小胶质细胞的稳态表型和抑制疾病相关表型是驻留 LXB 途径的潜在神经保护机制。
